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Invasive Procedure

Invasive Procedure

When Adams county fruit grower Jim Lerew first spotted the weird ring patterns on his peaches in 1998, he didn't know what to think.

"This was fruit that had been culled at the packing house," remembers John Halbrendt. "Nobody would buy it."

Lerew took some samples to Penn State's Fruit Research and Extension Center in nearby Biglerville, where Halbrendt works as a plant pathologist. "We didn't know what it was," Halbrendt says. "There are so many things it could have been. Some diseases mimic other diseases, or nutritional problems, or mineral deficiencies. . . You can get a frost at the wrong time and it creates symptoms that happen once and you never see them again. So the first time he brought it in, nobody got excited.

"But then it showed up a second year. Not in a lot of trees, so even [Lerew] wasn't too alarmed, but he wanted to find out what was wrong, so he could correct it."

Finally, toward the end of the growing season, Lerew took some of the suspect peaches to a fruit-growers meeting at Rutgers University, and showed them to Jerry Frecon, an extension specialist who had studied for a time in France. "[Frecon] said, 'If I didn't know better, I'd say that was plum pox.' It was a casual remark like that," Halbrendt remembers.

That was September 9, 1999. On September 23, Ruth Welliver, plant virologist for the Pennsylvania Department of Agriculture, visited the Lerew Brothers orchard in Gardners, near Gettysburg. She took leaf samples back to her lab in Harrisburg and tested them against antibodies that react to the large family of pathogens known as potyviruses. A week later she had a positive result. "There is only one potyvirus that affects stone fruits," Welliver said soon after. "So this was not a good sign."

Welliver sent a picture of the problem peaches to colleague Laurene Levy at the U.S. Department of Agriculture's laboratory in Beltsville, Maryland. When Levy saw those ring patterns, she told a Science News reporter later, "I just about died." Subsequent tests by USDA scientists confirmed the presence of plum pox virus.

"Very quickly we had a group of volunteers organized to go out to the neighboring orchards and take samples for testing," Halbrendt says. It was the end of the growing season, and Welliver needed leaf tissue to run her tests.

By first frost, she had her results. A total of 18 orchard blocks, all within the same three-mile area, had turned up plum-pox infected trees. Disturbingly, only two of these blocks had trees that showed any symptoms.

The Pennsylvania Department of Agriculture quickly imposed a quarantine on the two rural townships affected to prevent movement of infected plant material and spread of the virus: No Prunus seedlings, buds or other living tissues could be shipped out of the area. In December, an emergency meeting at the Penn State extension center in Biglerville drew researchers and growers from across the U.S. and Europe. Sam Hayes, Pennsylvania's Secretary of Agriculture, pledged an aggressive response.

"It was only a couple of miles from here that one of the great battles of the Civil War was waged," Hayes said. "We're about to wage another one."

Plum pox doesn't hurt humans. But it is one of the most devastating diseases that can hit the plant species Prunus, which include peaches, plums, apricots, nectarines, almonds, and cherries. First reported in 1915, in Bulgaria, the plum pox virus gradually spread throughout Europe, where it has become the scourge of the stone-fruit industry. Estimates are that over 100 million European trees are infected with the disease, which causes severe cosmetic damage to fruit, making it unsalable, drastically reduces yields, and may eventually kill the tree. More recently, plum pox has spread along the Mediterranean basin and as far as Egypt and Syria. In 1992, it was discovered in Chile. Before the outbreak five years ago in Adams County, however, it had never been seen in North America.

The immediate fear, then, was that the virus would spread: not just through Adams County, where peaches are an important pillar of a local economy that depends heavily on fruit trees, but also to threaten Pennsylvania's sizeable stone-fruit industry, the country's fourth largest, with an annual value of $25 million. Beyond that, there was real concern that plum pox, if not checked, would make its way clear across the U.S., crippling an industry worth some $1.8 billion.

The immediate reaction, therefore, was to search and destroy. "We must circle this virus and do everything humanly and scientifically possible to confront it," Secretary Hayes said. Soon after the ground thawed in the spring of 2000, several hundred acres of infected peach trees were bulldozed, with a three-year moratorium placed on replanting. A massive survey effort was commenced to check for plum pox in all commercial orchards and nurseries across the state.

Even before that, however, Penn State's College of Agricultural Sciences and Cooperative Extension service had swung into action. Plant pathologists Jim Travis and Herb Cole organized a series of educational meetings—for growers, but also for researchers and extension agents who had never worked with the disease. Penn State experts appeared on radio and television programs around the state. Agriculture communications teams, working closely with Travis, developed pamphlets, a video, and a Web site that became a clearinghouse for plum pox information.

"There was a lot of concern among the growers, " Halbrendt remembers. "I don't think anyone's panicking," Jim Lerew told an interviewer. "Fruit growers deal with problems all the time—freezes, hailstorms, droughts, too much rain at the wrong time—this is just another problem to deal with." Still, Jim Lott, another Adams County grower, acknowledged, "the more I learn about it, the scarier it gets." Un-checked, Lerew admitted, plum pox would put Pennsylvania out of the peach business.Experts warned of a possible domino effect. In Adams county, peaches fit neatly into a balanced system with apples, the crop the region is better known for. "The breakdown is probably 75 percent apples, 25 percent peaches," Penn State plant pathologist Fred Gildow estimates. But peaches bring much more than apples do at market. And crop diversity is crucial to efficient harvesting. "We depend a lot on migrant workers down here," Halbrendt explains. "You want to spread out your harvest season so you can get everything picked. Peaches come in early, so you can get your pickers to come in during the peach season, and then stay on through the apple season. You can give them a full season's work. Otherwise, they're not going to want to come."

Howard Nuernberger

After plum pox was detected in Adams County in late 1999, several hundred acres of infected peach trees had to be bulldozed. Penn State researchers and extension specialists have played important roles in subsequent efforts to contain the outbreak, which posed a serious threat to the stone fruit industry.

By the time European plant scientists knew what they were dealing with decades ago, plum pox virus (known there by its Slavic name, "sharka") was too entrenched to eradicate. Because the Pennsylvania outbreak appeared to be an isolated incident, experts hoped they had caught it early enough to get rid of the disease entirely.
infected peach trees

Several hundred acres of infected peach trees had to be bulldozed.

They were cautiously optimistic. "If there is a chance, now is the time," Halbrendt said at that first emergency meeting. "We're going to have to be ruthless."

"The key is the growers," Ruth Welliver said. "We have to have grower commitment to cleaning it up, to looking at their orchards and communicating what they see."

By all accounts, that commitment was strong from the beginning. But it was certainly helped when the Pennsylvania legislature authorized an indemnification program aimed at providing growers with fair compensation for the losses they would incur if their lands turned out to be infected. The work of Penn State economist Jayson Harper and others to detail plum pox's potential impact on the state, along with strong support from the Department of Agriculture, helped to get the bill passed, and also to spur the federal government to institute a similar program. Currently, the federal government bears 85 percent of the indemnification cost, and Pennsylvania the remaining 15 percent.

As important to the goal of eradication was a massive and well-coordinated survey effort. In the spring of 2000, survey crews set out to sample and test every peach orchard in the state of Pennsylvania. Orchards in Adams and surrounding counties were tested twice. In areas with infected orchards, volunteers conducted door-to-door surveys to locate backyard fruit trees for testing.

Collectors walked each orchard, "taking leaves from every fourth tree," Gildow says. Because the leaves often show no outward signs of the virus, they had to be tested serologically. Back at her Department of Agriculture lab, Welliver and her staff would pulverize the leaves and then subject them to Enzyme-Linked Immunosorbent Assay, or ELISA, a standard method of testing for plant diseases. An ELISA set up for plum pox contains antibodies specific to the virus: It reacts in the presence of proteins present in infected leaves. Samples testing positive were then sent on to the USDA's lab at Beltsville, Maryland, where they were double-checked via polymerase chain reaction, or PCR, which shows up the presence of viral RNA.

"Wherever a positive was confirmed, the PDA survey crews would go back and do follow-up testing," Gildow says. Once infected orchards were identified, they had to be immediately removed and burned—as did all surrounding trees to a distance of 500 meters. That distance, Gildow explains, is safely beyond the normal flight range of the aphids that carry plum pox from tree to tree.

That first year, over 65,000 samples were tested, and 399 turned up as positives for plum pox virus. Nine-hundred acres had to be destroyed.

At the same time, a national survey was being conducted by the USDA, testing orchards in all 35 peach-producing states. That task was made all the more urgent in June of 2000, when Canadian agriculture officials reported the existence of plum pox in Niagara Falls, Ontario

By the spring of 2002, authorities seemed to be getting a handle on the disease. The number of samples tested the previous summer had jumped to 140,000, and the number of positives fell dramatically, to 27. More importantly, the wider state and national surveys had turned up not a single positive result: No plum pox outside the original "hot zone" in Adams County. "That was a really good feeling," Welliver says, "to think that we had caught it in time."

But important questions remained. How did plum pox get to Pennsylvania? Where did it come from? How does it spread?

As an expert on plant virus transmission, Gildow already had some of the answers. "In nature," he explains, "potyviruses (the family of plant viruses to which plum pox belongs) are transmitted by aphids," tiny winged insects that feed in and colonize orchards and crop fields.

Aphids are sap-suckers: For their food, they fly from leaf to leaf, tapping into vascular tissue with a beak-like stylet, much the way a mosquito taps the blood of your arm. "The thing is, aphids aren't very smart," Gildow says. "They don't use their eyes or sense of smell to detect potential food hosts. To determine whether a leaf is one they'll like, they land and taste. That's when the virus gets spread, when they're hopping from tree to tree doing these test probes."

The virus can occur in high concentrations in leaf cells, he explains. When an aphid samples an infected cell, virus is drawn into the stylet. Although the aphid itself will not be infected, the virus can remain potent in its stylet for a couple of hours. If, in that time, the aphid lands on a healthy plant and takes a taste, some of that live virus is transferred and the virus can be spread.

It's a pretty inefficient means of transmission: A French study of apricot orchards showed that only one in a thousand aphids is likely to pass plum pox along. Still, according to another study, up to 300,000 aphids may land on a tree in a given season. Sheer numbers, then, helped the aphid carry plum pox across the European continent, to the former Yugoslavia in 1935, Hungary in 1941, Germany in 1956, Poland and Russia in 1961, and France in 1970. "Spain was saved for a while by the Pyrenees," Gildow says. "Aphids couldn't cross over them." But plum pox finally crashed into Spain in 1984 by a far more insidious means: It was carried by human hands.

Trade in seedlings used as root stocks and in budwood is a staple of plant breeding, as new varieties are tried and old ones combined in the quest for the perfect tree. "Typically you would graft buds from good fruit varieties onto root stocks with other desirable characteristics," Gildow says. "Producing budwood is an industry in itself."

Most countries now place strict limits on this movement. Thus, Gildow says, "Any plant material entering the U.S. has to go through one of the USDA's two quarantine nurseries, either Beltsville, Maryland, or Prosser, Washington. You have to send the material to one of these nurseries, and they grow it, study it, test it for disease, and after two or three years, if it's clean—pathogen-free—they release it for nursery production and eventual sale."

USDA's regulatory arm, the Animal and Plant Health Inspection Service, tries to make sure nothing slips through this system. ("They're the people at the airport with the beagles," Gildow says.) But it's impossible to keep everything out. "You could take a couple of buds, put them in a damp paper towel and stick it in your pocket and bring them home and graft them." Since the infection is often present without symptoms, it would've been easy for an overzealous hobbyist to bring it in inadvertently. Alternatively, because the virus can remain latent and is extremely difficult to detect in some hosts, it may have entered the U.S. accidentally in infected Prunus ornamental or fruit seedlings and slipped through the system.

Infected buds or seedlings accidentally imported into the country are almost certainly how plum pox invaded North America, Gildow says. "Because it takes several years to see symptoms following infection, we think the introduction probably occurred eight or ten years ago."

A tight quarantine and an aggressive awareness campaign might stop infected budwood and root stock from leaving Adams County. But quarantines have little impact on the meanderings of aphids. To control the movement of plum pox at the local level would require learning exactly which aphids are responsible for its spread. As Gildow explains, there are reported to be about 10 aphid species that make their homes in North American orchards, with another dozen species routinely passing through on their way to or from other types of vegetation.

Because plum pox virus is one of 11 select plant pathogens identified under the U.S. Agricultural Bioterrorism Protection Act (see sidebar), it cannot legally be studied outside a secure biological containment facility. Penn State has no suitable facility, so Gildow does his work at the USDA's Foreign Diseases Containment Greenhouse at Fort Detrick, Maryland. There, over the last four years, he has been working in collaboration with USDA scientists Vern Damsteegt and Bill Schneider on studies of plum pox virus transmission.

"Of about 13 aphid species we tested from Pennsylvania orchards, Gildow reports, "we found four to be good vectors—that is, they transmitted the virus efficiently." The most efficient species was Myzus persicae, also known as the green peach aphid. But the most numerous in Pennsylvania orchards, by far, and therefore probably the main culprit, is the spirea aphid, Aphis spiraecola. "Both of these species are migrants, just passing through peach orchards during the growing season," Gildow says. "They blow in off other crops, or live on native plants or weeds in and around orchards. They're not going to colonize peach trees, but they will do test probes and feed on peach." The fact that both species are migrants and do not settle down and reproduce on peach means that pesticide sprays will not control them, Gildow adds. That makes quick eradication of infected trees even more important.

Gildow, Damsteegt, and Schneider have also found that the insects can pass virus from infected fruit to healthy trees—at least in the lab. This finding may prove especially important, since the current terms of quarantine do not forbid shipping infected fruit. "There's no proof that aphids in nature have picked up the virus from fruit and transmitted it to a tree," Gildow acknowledges. "But in a few cases we've found the virus in isolated trees, far from any other infection sites. And what these cases had in common was nearby cull piles—places where people have thrown reject fruit after packing or home use. What it suggests is another possible mode of transmission that needs to be looked at."

Another question that hung in the air was the possibility that plants other than Prunus might play a hidden role in plum pox's spread. Could aphids carry the virus to infect other nearby weeds or trees that might act as reservoirs—safe havens where the virus could survive and adapt to strike another day? If so, the task of eradication would become immensely more complicated.

"In the lab," Gildow says, "plum pox has a very large host range." Indeed, French plant virologists have identified 134 species of plants in 16 different families that can accommodate the virus. "But in the lab these plants are mechanically inoculated," he quickly adds. "You rub an abrasive on the leaf and then introduce the virus and see if it takes. Is that relevant in nature?" Damsteegt and Schneider, in collaboration with Gildow, are studying the plant host range of the virus in ornamental and wild Prunus species. When inoculated with aphids under greenhouse conditions, they have found the Pennsylvania isolates of plum pox virus capable of infecting most Prunus species tested, with the exception of sour cherry. "Many people may not be aware of the fact", says Gildow, "that many ornamental shrubs used for landscaping are Prunus species and that the number one hardwood tree in Pennsylvania is black cherry, another potential reservoir host of plum pox."

For the past four summers, John Halbrendt and a crew of helpers have been scouring the Adams County countryside in an effort to find out whether plum pox may have escaped the orchard and infected native plants or weeds which could hide the virus from the eradication effort.

"We go to areas adjacent to where heavily infected orchards had been," Halbrendt explains, "where there's been an opportunity for aphids to move back and forth between weeds [i.e., wild plants] and infected peach trees. We gather samples, identify them, and take them back to the lab and put them through ELISA." Last summer his team ran some 10,000 samples, representing 66 native species. "In one sense we're shooting in the dark," Halbrendt acknowledges. "But in another sense we can't ignore the possibility that it could be out there. You don't find what you're not looking for." So far, he reports, the news has been all good. Of a total of 37,000 samples tested over four seasons, they have found no infected weeds.

Halbrendt is also taking another approach: setting out "bait" plants and conducting surveillance on them. "These are herbaceous plants that have been identified as possible alternate hosts for the virus—I'm using pea, zinnia, African daisy, and red clover," he explains. "I germinate them in the greenhouse and take them out to sites in the quarantine area—again, these are sites adjacent to infected areas." After two weeks in the field—enough time for aphids to move in and feed—he brings the bait back to the lab, and tests it for plum pox. Again, after testing 9,000 plants, Halbrendt has seen no presence of the virus. (USDA personnel conducting a second surveillance program using peach trees as trap plants have had the same negative results.)

Is he willing, yet, to say that plum pox is not out there in the wild? "Well, you can't prove a negative," Halbrendt says. "The best we can do is keep piling up the data—the more we have the more confidence we can have in it. The number of samples we've run is still relatively small."

Even granting such a caveat, the concerted effort to deny plum pox's conquest of Pennsylvania—and of North America—appears to be working. There have been significant losses, to be sure. Some 1,400 acres, about a sixth of the stone-fruit acreage in the state, have had to be torn up. A number of Adams county orchards and nurseries have been hit pretty hard. Nationwide, an estimated $40 million has been spent so far in the eradication campaign. But the timely response appears to have stopped plum pox in its tracks. In 2003, Ruth Welliver reports, the Pennsylvania Department of Agriculture processed 200,000 stone-fruit samples, and turned up only 11 positives for plum pox, all within or near the original quarantine area. Even more importantly, the virus has not been spotted anywhere else in the U.S. The Ontario outbreak, while far more widespread at its outset, appears also to be under control.

This success, Gildow says, can be traced to a high degree of teamwork among the various agencies that came together to battle plum pox from the beginning. "There was no competition," he says. "This was an example of interagency cooperation that worked well."

Of particular importance, most agree, was the early intervention of the Pennsylvania Department of Agriculture, and a Secretary who had been sensitized by another recent emergency: the 1997 outbreak of Avian flu. "To have Sam Hayes on board, and realizing how important this was early on was critical," Halbrendt says. The current Secretary, Dennis C. Wolff, has also strongly backed the eradication program.

The fast and well-coordinated action of Penn State agricultural scientists and extension specialists was also critical. And Adams County growers, Halbrendt adds, "have been wonderful. They realized that to eradicate [the virus] would hurt now, but in the long run it would be better for everyone—and that if we didn't eradicate it, there would be a big black cloud over the state's stone-fruit industry from now on."

In 2002, the multi-agency team assembled to fight plum pox was officially recognized by U.S. Secretary of Agriculture Ann Veneman for effectively limiting the spread of plum pox "by building an inclusive team of university, state, federal, and agricultural industry personnel."

Potyviruses like plum pox are transmitted by tiny culprits

The kudos don't mean the fight is over, however. "It could be out there and we just haven't found it yet," Halbrendt says. The national survey, after three years with no trace of the disease, has narrowed to seven states: Pennsylvania, New Jersey, Maryland, New York, Virginia, Michigan, and California, says USDA's Don Albright, director of the national program. Likewise, Welliver says, state survey teams are "tightening the noose," sampling more intensely in a more and more concentrated area. In addition to orchards and nurseries, Albright's USDA teams set out last year to survey all 45,000 homeowner properties within the three-county quarantine area. One of those tests turned up a positive in a dwarf flowering almond tree—the first case of plum pox seen in an American ornamental.

Gildow has written: "As the eradication program continues and fewer infected trees remain, it will become even more important to identify every single infected tree. Elimination of 99 percent of the infected trees is not an option. Unless every infected tree is eliminated . . . plum pox virus epidemics can reoccur within a few years."

As an added precaution, in the wake of his fruit-transmission results, Gildow travels periodically to the port of Philadelphia to work with USDA-APHIS inspectors checking stone fruits arriving there from Chile, and testing them for possible PPV infection. "These fruits are arriving in winter," he says, "so I don't think it's very likely that they would play a major role in PPV spread. But there is always the remote possibility that some fruit could survive long enough to be discarded in cull piles, and the state has asked us to check on this."

The long-term solution, Halbrendt and Gildow agree, is to develop stone fruits that are virus-resistant, whether by traditional plant breeding, or by genetic engineering. USDA researchers have already developed a plum-pox resistant plum.

For now, however, plum pox—by all estimates—is at least contained. "That word seems a little weak, but I guess it's fair," Welliver says. "Contained, and hopefully on its way out."

"We have knocked it way back," Gildow concurs. "Now we need to be vigilant for the next five years. If we can do that, we will have saved the industry."